This project will generate and validate fourteen strains of mice with fully functional fluorescent mouse nicotinic receptor (nAChR) subunits. These strains will be constructed using the proven and reasonably efficient method of exon replacement via homologous recombination in embryonic stem cells, leading to "knock-in mice". In each case, one mGFP and one mCherry allele will be generated, so that strains can be crossed to study receptor assembly with Foerster resonance energy transfer. Fluorescent proteins (FPs) have been successfully integrated into the M3-M4 intracellular loop of most of these subunits (the final two will be verified before funding starts), and these fluorescent subunits are fully functional and correctly targeted in mammalian cell lines. The Caltech group will immediately construct targeting vectors for the alpha3, alpha4, alpha5, alpha6, beta2, beta3, and beta4 FP-labeled subunits. The project will generate embryonic stem cells harboring these subunits, then generate knock-in mice. Further experiments will validate that these strains display nAChRs whose key parameters of expression and function are within a factor of two of the wild type level. Thus, at Boulder autoradiographic and synaptosome-based experiments will be performed on the initial lines to verify faithful expression and function;at Caltech, cellular-level fluorescence quantification will also verify faithful expression and gene dose dependence. Simultaneously, the project will begin to generate congenic strains on the C57Bl/6 background. These mice will be valuable for future studies, for two key reasons which bear on multiple biomedical disciplines. First, evidence is emerging that changes in the level and composition of the nAChRs themselves underlie some components of the responses to chronic exposure to nicotine during nicotine addiction. Changes in the level of the receptors themselves are also thought to underlie two inadvertent therapeutic effects of tobacco use: the inverse correlation between a person's history of tobacco use and his risk of Parkinson's disease (PD), and the seizure-suppressing effects of nicotine use in autosomal dominant nocturnal frontal lobe epilepsy. Second, SNPs found in two clusters of nAChR subunit genes, and in two other nAChR subunit genes, are associated with nicotine dependence, number of cigarettes smoked per day, "pleasurable buzz" elicited by smoking, age of tobacco and alcohol initiation, early subjective response to tobacco, "dizziness" after the first few cigarettes, and lung cancer. Other studies have linked one or both clusters with alcohol and cocaine dependence. Smoking is also clearly associated with chronic obstructive pulmonary disease. Time lines and decision points for this research are established, as are procedures for distribution to the research community. It is likely that congenic strains will be available less than a year after the project terminates. PUBLIC HEALTH RELEVANCE: The mice generated in this project will be valuable for future studies, for reasons which bear on several diseases. First, evidence is emerging that changes in the level and composition of the nicotine receptors themselves underlie some components of the responses to chronic exposure to nicotine during nicotine addiction. Changes in the level of the receptors themselves are also thought to underlie two inadvertent therapeutic effects of tobacco use: the inverse correlation between a person's history of smoking and his risk of Parkinson's disease, and the seizure-suppressing effects of nicotine use in autosomal dominant nocturnal frontal lobe epilepsy. Second, variations found in two clusters of receptor subunit genes, and in two other receptor subunit genes, are associated with nicotine dependence;number of cigarettes smoked per day;"pleasurable buzz" elicited by smoking;age of tobacco and alcohol initiation;early subjective response to tobacco;and lung cancer. Other studies have linked one or both clusters with alcohol and cocaine dependence. Smoking is also clearly associated with chronic obstructive pulmonary disease.